Molecular analysis of different classes of RNA molecules from formalin-fixed paraffin-embedded autoptic tissues: a pilot study.
Author(s): Muciaccia B, Vico C, Aromatario M, Fazi F, Cecchi R
Publication: Int J Legal Med, 2015, Vol. 129, Page 11-21
PubMed ID: 25135750 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effect of postmortem interval (PMI) on the yield and integrity of RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and to assess its suitability for qRT-PCR analysis of constitutively expressed mRNA and small RNA transcripts. RNA yield, quality and qRT-PCR results were also compared in a single pair of case-matched FFPE and Bouin’s fixed, paraffin-embedded biopsy specimens.
Conclusion of Paper
RNA yield and integrity (assessed by RNA integrity number, RIN) were not affected by PMI (18-96 h) in the 4 postmortem cases examined , but RINs were low for all specimens. The small RNA fraction (6-150 nt) increased with PMI, suggesting an increase in degradation over time. qRT-PCR analysis of mRNA was not successful in postmortem tissue specimens, although small RNAs were successfully amplified in the majority of tissues subjected to a PMI of 72 h at 4°C or less. When the differentially fixed pair of biopsies was compared, RNA yield was higher in the formalin-fixed than Bouin’s- fixed specimen. Although RINs were comparable between the two specimens, the formalin-fixed specimen had longer RNA fragments, and a smaller percentage of small RNA compared to the Bouin’s-fixed specimen, indicating less RNA degradation. While qRT-PCR analysis of small RNAs were successful in both specimens, qRT-PCR analysis of mRNA was only successful in the formalin-fixed specimen.
Studies
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Study Purpose
Study Purpose
The purpose of this study was to determine the effect of postmortem interval (PMI) on RNA yield, integrity and the successful detection of mRNA and small RNA transcripts by qRT-PCR. Tissue biospecimens from liver, lung, kidney, and myocardium were collected at autopsy from each of four individuals that experienced an accidental death but were otherwise healthy and for which PMI was recorded. Brain specimens were also collected from two of the four cases, and three of the four cases were maintained under refrigerated conditions (4°C) immediately after death until autopsy. Details of the condition of the remaining case prior to autopsy were not specified although tissue putrification was observed upon autopsy. Each case differed for age at death (22, 29, 71, and 80 y) and PMI (18, 72, and 96 h at 4°C, 65 h at an unspecified temperature). RNA was extracted from five 10 µm-thick paraffin sections (after the first 2-3 sections were discarded) using Qiagen's miRNeasy FFPE kit. RNA yield and purity were assessed by NanoDrop spectrophotometry, and RNA quality and integrity were assessed using an Agilent Bioanalyzer. The fraction of small RNA (6 to 150 nt) was determined using a Small RNA kit in conjunction with a bioanlyzer. RNA transcript levels of beta-actin, histone H3, GAPDH, and small RNAs RNU6-2 and Hs-miR-21-5p were quantified by qRT-PCR.
Summary of Findings:
RNA yield differed among individual cases and among tissue types (ranging from 26.8 to 593.0 ng/µl), although there was no observable pattern with regards to tissue type, PMI or RNA integrity. While RIN was not provided for each tissue type and PMI (case), the authors note that RNA isolated from postmortem FFPE specimens was of low quality, with tissues subjected to the shortest PMI investigated (18 h at 4°C) generating a mean RIN of 2.30 (ranging from 1.80 to 4.20). The majority of postmortem specimens had electropherogram peaks at 60-80 nt, indicating RNA degradation, but the authors note the percentage of small RNA increased with PMI.. To illustrate, the small RNA fraction isolated from lung specimens increased from 18% at a PMI of 18 h, to 38% at a PMI of 72 h, to 40% at a PMI of 96 h, although it’s important to note specimens were not case-matched. While the magnitude of change differed among tissue types, the same trend was observed, with lung specimens displaying both the smallest small RNA fraction and the smallest change over the PMI investigated, while liver displayed the greatest changes. Severe RNA degradation, as determined from high percentages of small RNA (49 to 56%) were also observed among the putrefied tissue specimens isolated from a single case subjected to a PMI of 65 h under uncontrolled conditions. When tissue specimens collected from the case subjected to a PMI of 18 h at 4°C were assessed, amplification results for beta-actin, GAPDH, and histone H3 were all below the quality threshold for all tissue types. Amplification of two representative small RNAs, RNU6-2 and miR-21, by qRT-PCR was successful in all tissue specimens at all PMIs with the exception of 72 h PMI liver and kidney specimens and 96 h PMI liver, kidney, and myocardium specimens (for which miR-21 levels were below the quality threshold). For putrified tissue specimens, qRT-PCR analysis of miR-21 was successful for all tissue specimens with the exception of kidney, while RNU6-2 analysis was below the quality threshold for all tissue specimens.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
- Normal
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Postmortem interval 18 h
72 h
96 h
Real-time qRT-PCR Specific Targeted nucleic acid Beta-actin
GAPDH
Histone H3
RNU6-2
miR-21
Biospecimen Acquisition Biospecimen location Brain
Liver
Lung
Kidney
Myocardium
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Study Purpose
This study compared yield, integrity and qRT-PCR success of RNA isolated from two testicular biopsy specimens collected from the same patient that were fixed in either 10% neutral buffered formalin or Bouin’s fixative prior to processing and paraffin embedding. Additional information on patient diagnosis, and fixation and processing conditions and durations were not provided. RNA was extracted from five 10 µm-thick paraffin sections (after the first 2-3 sections were discarded) using Qiagen's miRNeasy FFPE kit. RNA yield and purity were assessed by NanoDrop spectrophotometry, and RNA quality and integrity were assessed using an Agilent Bioanalyzer. The fraction of small RNA (6 to 150 nt) was determined using a Small RNA kit in conjunction with a bioanalyzer. RNA transcript levels of beta-actin, histone H3, GAPDH, and small RNAs RNU6-2 and Hs-miR-21-5p were quantified by qRT-PCR.
Summary of Findings:
RNA yield was higher in the testicular biopsy specimen fixed in formalin compared to the specimen fixed in Bouin’s fixative (176 versus 84.1 ng/µl, respectively). While RINs were comparable (2.20 versus 2.40, respectively), the formalin-fixed specimen had longer RNA fragments (> 1,000 nt) and a smaller small RNA fraction (10-40 nt; 15 versus 36%, respectively) than the Bouin’s-fixed specimen. Analysis of beta-actin, GAPDH, and histone H3 transcript by qRT-PCR was only successful for the formalin-fixed specimen, while analysis of small RNAs (RNU6-2 and miR-21) was successful in both the formalin- and Bouin’s-fixed specimen.
Biospecimens
Preservative Types
- Other Preservative
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Bouin's fixative